Covalent modification of abnormal prion protein

ABSTRACT

A prion-physiological structure and associated method of formation. A provided abnormal prion has a transforming power over a normal prion to convert the abnormal prion into defective prion that mimics the abnormal prion. A linker molecule is then bonded to the abnormal prion, wherein a polymer that is covalently attached to the linker molecule facilitates formation of a polymerized abnormal prion that does not have the transforming power over the normal prion.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention relates to covalent modification ofabnormal prions for protecting against transmission of prion disease.

[0003] 2. Related Art

[0004] Prions are proteins that occur in the brains of all mammalsstudied to date. Although the normal function of a prion protein(“prion”) is not well understood, recent research on mice lacking theprion protein gene (which encodes the prion protein) suggests that theprion protein protects the brain against dementia and other degenerativeproblems associated with old age. Such a prion is a “normal prion.”

[0005] The normal prion protein (PrP) is a glycoprotein constitutivelyexpressed on the neuronal cell surfaces of mammals. The normal prionprotein can be altered, however, as a consequence of genetic mutationsor induced changes in protein shape and structure (conformationalchanges). Indeed, such altered or “abnormal” prion proteins (also calleddefective prions, infective prions, distorted prions, etc.) areimplicated in the pathogenesis of a number of inheritable and infectiousspongiform encephalopathies in human and nonhuman animals (see FIG. 6for both genetic and non-genetic modes of transmission). As infectiousdisease agents, the abnormal protein is transferred from one animal toanother via multiple routes. In humans, Creutzfeldt-Jakob Disease (CJD)has been transmitted via blood transfusion (Iatrognic transmission) anda bovine derived disease (CJD-New Variant) has been though to betransmitted via the consumption of meat obtained from cattle infectedwith Bovine Spongiform Encephalopathy (BSE). Although some abnormalprions may be transmitted genetically, non-genetic modes of transmissionare possible.

[0006] The concept of a protein as an infectious disease agent was firstproposed by Prusiner in the early 1980's (Prusiner, 1982). Since theinitial proposal of infectious proteins was made, the molecular/geneticbasis of the some human prion diseases have been characterized as shownin FIG. 6. In addition, animals models of prion diseases have also beensuccessfully developed.

[0007] Noteworthy aspects of diseases listed in FIG. 12 are as follows.CJD occurs when codon 129 is homozygous for valine or heterozygous formethionine and valine. Fatal Familial Insomnia (FFI) occurs when codonis 129 is homozygous for methionine. Codon 102, in relation toGerstmann-Straussler Syndrome (GSS), produces a prion protein thataccumulates in the endoplasmic reticulum. Kuru, found in the Fore tribeof New Guinea, is spread by ritual cannibalism in which mourners eat thebrains of their dead relatives, a practice that has since ceased. Forthe non-human diseases of Scrapie, BSE, Transmissible MinkEncephalopathy (TME), and Chronic Wasting Disease (CWD) of Deer, a cleargenetic predisposition is present but may not be sufficient; and someevidence suggests that an environmental agent is necessary to induce atleast some genetic versions of the disease. BSE is believed to haveresulted from feeding healthy cattle a processed form of sheep, whereinthe sheep had died from the related disease of Scrapie.

[0008] Abnormal prions transform, via conformational changes, any normalprions they encounter into copies of themselves, as illustrated inFIG. 1. In FIG. 1, an abnormal prion 12 in a receptive host animalinteracts with a normal prion 10 in the animal, resulting in a priontransformation process 14 which results in conversion of the normalprion 10 into an abnormal prion 16. The abnormal prion 12 acts as atemplate for the aforementioned transformation of the normal prion 10such that the normal prion 10, when transformed to the abnormal prion16, assumes the conformational shape of the introduced abnormal prion12. The representation of the normal prion 10 and abnormal prion 12 inFIG. 1 is a symbolic representation of a normal prion and an abnormalprion, respectively, and does not depict or suggest the geometry ofnormal and abnormal prions. A normal prion includes strands of a helicalshape that is called an “alpha helix” and such strands appear twistedinto a spiral. An abnormal prion includes both helical strands andstrands arranged in a planar shape called a “beta-sheet.” Thetransformation process 14 of FIG. 1 converts the alpha-helix structureof the normal prion 10 into the beta-sheet structure of the abnormalprion 12.

[0009] Assuming that the transformation process 14 of FIG. 1 occurs inthe brain of a host animal, the transformation process 14 continues likea chain reaction and eventually generates enough abnormal prions tocause destruction of brain neurons, resulting in an inflammatorycondition in the brain that is sponge-like in appearance. Hence,abnormal prions cause brain destruction. Unfortunately, there are noknown cures for prion diseases.

[0010] Since prion disease, for which there is no known cure, can betransmitted between humans, between non-human animals, and fromnon-human animals to humans, there are high health and economic costsrelating to prion disease. Accordingly, there is a need for protectingagainst transmission of prion disease.

SUMMARY OF THE INVENTION

[0011] The present invention provides a method for forming aprion-physiological structure, comprising:

[0012] providing an abnormal prion that has a transforming power over anormal prion; and

[0013] covalently bonding a linker molecule to the abnormal prion,wherein a polymer is covalently attached to the linker molecule to forma polymerized abnormal prion that does not have the transforming powerover the normal prion.

[0014] The present invention provides a prion-physiological structure,comprising:

[0015] an abnormal prion that has a transforming power over a normalprion; and

[0016] a linker molecule covalently bonded to the abnormal prion,wherein a polymer is covalently attached to the linker molecule to forma polymerized abnormal prion that does not have the transforming powerover the normal prion.

[0017] The present invention provides a prion-physiological structure,comprising:

[0018] an abnormal prion that has a transforming power over a normalprion; and

[0019] means for covalently bonding a linker molecule to the abnormalprion, wherein a polymer is covalently attached to the linker moleculeto form a polymerized abnormal prion, and wherein the polymer has a longchain length that inactivates the transforming power.

[0020] The present invention protects against transmission of priondisease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 depicts a transformation of a normal prion into an abnormalprion, in accordance with the related art.

[0022]FIG. 2 depicts how polymerization of an abnormal prion preventstransformation of a normal prion into an abnormal prion, in accordancewith the embodiments of the present invention.

[0023]FIG. 3 depicts introduction of polymerized abnormal prion into ananimal, in accordance with embodiments of the present invention.

[0024]FIG. 4 depicts an exemplary chemistry of coupling a polymeratedlinker chemical to protein in an abnormal prion, in accordance withembodiments of the present invention.

[0025]FIG. 5 lists exemplary polymeric linker compounds and associatedprotein or carbohydrate targets that can be reacted with the exemplarypolymeric linker compounds, for use in conjunction with FIGS. 2 and 3,and in accordance with embodiments of the present invention.

[0026]FIG. 6 lists examples of human and animal diseases caused byabnormal prion proteins, in accordance with the related art.

[0027]FIG. 7 depicts loss of mobility of red blood cells in an electricfield, resulting from pegylation of the red blood cells.

[0028]FIG. 8 depicts loss of red blood cell Rouleaux Formation,resulting from pegylation of the red blood cells.

[0029]FIG. 9 depicts non-sedimentation of red blood cells, resultingfrom pegylation of the red blood cells.

DETAILED DESCRIPTION OF THE INVENTION

[0030] An abnormal prion that is able to transform a normal prion intoan abnormal prion, as described supra in conjunction with FIG. 1, issaid to have a “transforming power” over the normal prion. The presentinvention discloses a method and structure for modifying an abnormalprion in a way that the abnormal prion so modified no longer has thetransforming power over the normal prion.

[0031]FIG. 2 illustrates how the present invention preventstransformation of a normal prion into an abnormal prion (which removesthe transforming power from the abnormal prion), in accordance with theembodiments of the present invention. In FIG. 2, a normal prion protein20 is proximate an abnormal prion protein 22 that has been covalentlybonded to a polymerated linker chemical 25 to form a polymerizedabnormal prion 24. The polymerized abnormal prion 24 comprises theabnormal prion protein 22 covalently bonded to the polymerated linkerchemical 25. The polymerated linker chemical 25 includes a linkermolecule 26 with a covalently attached polymer 27. The polymeratedlinker chemical 25 is said to represent an activated form of the polymer27. For example, if the polymer is methylpolyethylene glycol (mPEG),then “activated mPEG” is exemplified by having mPEG covalently bonded tothe linker molecule of cyanuric chloride. As another example, if thepolymer is polyethylene glycol (PEG), then then “activated PEG” isexemplified by having PEG covalently bonded to the linker molecule ofcyanuric chloride. The linker molecule 26 is covalently bonded to aprotein or carbohydrate of the abnormal prion 22. The covalent linkingof the linker molecule 26 to a protein may include a covalent linking ofthe linker molecule 26 to an amino acid in the protein or to asulfhydryl group in the protein. The polymer 27 has a “long chainlength;” i.e., a chain length that is of sufficient magnitude to fillthe space around itself to create the blocker envelope 28. The blockerenvelope 28 constitutes a barrier that prevents the abnormal prion 22from interacting with the normal prion 20. In addition, the polymer 27within the blocker envelope 28 inhibits, by steric hindrance,interaction between the abnormal prion 22 and the normal prion 20.Additionally, the polymer 27 may be highly hydrophillic so as to createa hydration zone around itself to alternatively create the blockerenvelope 28. Inasmuch as the abnormal prion 22 and the normal prion 20would interact via a charge-charge coupling mechanism, the hydrationzone encompassed by the blocker envelope 28 effectively camouflagesmolecular charge sites at the abnormal prion 22 and thus prevents theelectrical interactions that would cause the normal prion 20 to betransformed into an abnormal prion. Thus in FIG. 2, the normal prion 20is not transformed into an abnormal prion, because the polymerizedabnormal prion 24 does not have the transforming power over the normalprion 20.

[0032] The blocker envelope 28 may be generated by the polymer 27 by anysuitable method for bonding the polymerated linker chemical 25 toabnormal prion 22, such as, inter alia, spraying the polymerated linkerchemical 25 onto the abnormal prion 22, immersing the abnormal prion 22into a liquid medium that includes the polymerated linker chemical 25,reacting the polymerated linker chemical 25 with the abnormal prion 22with further processing to create a pill that includes the polymeratedlinker chemical 25 enveloped around the abnormal prion 22, etc.

[0033] In FIG. 2, the interaction (or lack thereof) between the normalprion 20 and the abnormal prion 22 takes place in an environment 19. Asan example, the environment 19 may comprise proteinaceous matter suchas, inter alia, blood albumin intended for subsequent introduction intoa human or non-human animal. Unfortunately, present technology is unableto test for abnormal prions in blood albumin. Thus, by removing thetransforming power of any abnormal prions that may be present in theblood albumin, the present invention enables said subsequentintroduction of the blood albumen into a human or non-human animal to beaccomplished with little or no risk of infecting the animal with priondisease. As another example, the environment 19 may comprise the brainof the human or non-human animal into which the polymerized abnormalprion 24 has been introduced.

[0034]FIG. 3 depicts introduction of polymerized abnormal prion 34 intoan animal 60, in accordance with the embodiments of the presentinvention. The polymerized abnormal prion 34 includes a polymeratedblocker chemical 35 covalently bonded to an abnormal prion 32. Thepolymerated blocker chemical 35 comprises a polymer 37 covalentlyattached to a linker molecule 36. The polymer 37 includes a blockerenvelope 38.

[0035] The animal 60 may be a human animal (e.g., a human being or afetus) or a veterinary animal. A veterinary animal is a non-human animalof any kind such as, inter alia, a domestic animal (e.g., dog, cat,etc.), a farm animal (cow, sheep, pig, etc.), a wild animal (e.g., adeer, fox, etc.), a laboratory animal (e.g., mouse, rat, monkey, etc.),an aquatic animal (e.g., a fish, turtle, etc.), etc. The animal 60 hasan interior 64 which includes a brain 65 that contains a normal prion66.

[0036] The polymerized abnormal prion 34 enters the animal 60 through anentry 63. The entry 63 denotes any entry into the animal 60 into which,or through which, the polymerized abnormal prion 34 may enter the animal60. The polymerized abnormal prion 34 may enter the animal 60 throughthe entry 63 by any method or mechanism that is known to one of ordinaryskill in the art for introducing a prion into an animal (or any possiblemethod or mechanism if said entry is unintentional) such that thepolymerized abnormal prion 34 that enters the animal 60 is subsequentlytransported into the brain 65 of the animal 60 through any physiologicalpathway (e.g., a vascular pathway) 67. Entry of the polymerized abnormalprion 34 into the animal 60 through the entry 63 is illustrated by thefollowing examples. As a first example, the entry 63 may be a mouth intowhich the polymerized abnormal prion 34 enters in pill, liquid, or sprayform, or by food consumption (e.g., beef bouillon cubes). As a secondexample, the entry 63 may be a nose into which the polymerized abnormalprion 34 enters by a nasal spray. As a third example, the entry 63 maybe a blood vessel into which the polymerized abnormal prion 34 enters bytransfusion or injection. As a fourth example, the entry 63 may be amuscle into which the polymerized abnormal prion 34 enters by needleinjection. As a fifth example, the entry 63 may be a vagina (if theanimal 60 is female) into which the polymerized abnormal prion 34 entersvia use of a syringe (e.g., use of prion contaminated cattle semen).

[0037] There are various circumstances under which the polymerizedabnormal prion 34 may enter into the animal 60, including, inter alia,the following circumstances. A first circumstance includes introducing ablood product (e.g., blood albumin from blood plasma) into the animal60, where it has not (or cannot) be determined whether the blood productcontains any prions. A second circumstance includes introducing a foodproduct into the animal, where the food product has been previouslytreated with a polymerated linker chemical to covalently bond with anyabnormal prions contained within the food product. A third circumstanceincludes introducing the polymerized abnormal prion 34 into a non-humananimal for purposes of research relating to interactions between thepolymerized abnormal prion 34 and the normal prion 66 in the brain 65.

[0038]FIGS. 2 and 3 show “prion-physiological structures.” Aprion-physiological structure is defined herein as an organic structurethat includes a prion, together with any animal that comprises the prionand with any chemical that is covalently bonded to the prion.

[0039]FIG. 4 illustrates an exemplary chemistry of coupling thepolymerated linker chemical (as depicted in FIG. 2 or FIG. 3) to aprion, in accordance with embodiments of the present invention. In FIG.4, two chemical reactions are illustrated. In the first chemicalreaction shown in FIG. 4, a polymer 80 reacts with a linker molecule 81to form a polymeric linker chemical (PLC) 82 in which the polymer 80 iscovalently bonded to the linker molecule 81. Specifically in FIG. 4, thepolymer 80 is methoxypolyethylene glycol (mPEG) having the chemicalstructure of CH₃(—O—CH₂-CH₂)_(n)—OH wherein n≧2. The linker molecule 81is an alkyl halide (namely, cyanuric acid) and the resultant PLC 82 is2-O-mPEG-4,6-dichloro-s-triazine. In the first chemical reaction, thehydroxyl group (OH³¹ ) is a nucleophile that reacts generally with analkyl halide (specifically, cyanuric chloride), resulting indisplacement and release of the chlorine ion (CL⁻) in position 2 of thecyanuric chloride triazine ring as well as release of the hydrogen ion(H⁻) from the hydroxy group of the mPEG. The first chemical reaction maybe implemented in any manner known to one of ordinary skill in the artsuch as in, inter alia, anhydrous benzene at a temperature of about 25°C. Formation of the PLC 82 of 2-O-mPEG-4,6-dichloro-s-triazine iswell-known in the art and may be obtained commercially.

[0040] In the second chemical reaction shown in FIG. 4, a protein 83reacts with the PLC 82 to form a protein-polymer complex 84.Specifically in FIG. 4, the protein 83 includes lysine, whereinH₃N⁺—(CH₂)₄ is a portion of the lysine that reacts with the PLC 82, andwherein X represents a remaining portion of the protein 83 including aremaining portion of the lysine. The remaining portion of the lysine hasa carbon atom covalently bonded to H, H₃N⁺, and a carboxyl group. Asshown in FIG. 4, a hydrolysis of the chlorine in position 4 of thecyanuric chloride triazine ring has replaced said chlorine in position 4with the H₃N⁺—(CH₂)₄ portion of the lysine of the protein 83, to formthe protein-polymer complex 84. Specifically in FIG. 4, theprotein-polymer complex 84 is 2-O-mPEG-4-Y-6-chloro-s-triazine, whereinY is the protein H₃N⁺—(CH₂)₄—X. More generally, FIG. 4 shows generationof a PEG-conjugated protein with attachment of an activated PEG (e.g.,the PLC 82) to an e-amino group (e.g., the lysine or another amino acidsuch as arginine). The second chemical reaction may be implemented in analkaline phosphate buffer (e.g., 50 mM of K₂HPO₄ and 105 mM of NaCl,wherein mM denotes millimoles). The second reaction can be efficientlyaccomplished in a wide range of media including, inter alia, saline,phosphate buffered saline, blood plasma, blood serum, albumin containingbuffers, Hanks Balanced Salt Solution (HBSS),N-[2-hydroxyethyl]piperazine-N′-2-ethanesulfonic acid (“HEPES”), RoswellPark Memorial Institute 1640 (“RPMI 1640”), etc.

[0041] Time and temperature for performing the second reaction are veryflexible. For example, a reaction between mPEG and an amino acid of aprion may be accomplished in 4 minutes or longer at 4° C. if the pH isabout 9. If the pH is lower (e.g., about 8), the reaction may proceed atroom temperature for a longer period (e.g., 60 minutes or longer) sothat the virus is stressed by temperature and not stressed by harshalkaline conditions. As to pH, it is useful to have a pH of about 8 whenreacting mPEG with lysine. When reacting mPEG with a prion, weaklyacidic to alkaline conditions should be used with a representative pHrange of about 6.0 to about 9.0.

[0042] Effective doses of the PLC in the second reaction depend onseveral variables, including: linker chemistry, the polymer being used,surface area of prion surfaces being modified, etc.

[0043] It should be noted that the chlorine in position 6 of thecyanuric chloride triazine ring is quite unreactive and thus unavailableto react with either an amino acid or with a second polymerated linkerchemical.

[0044]FIG. 4 illustrates a mechanism of the covalent attachment of thepolymerated linker chemical of cyanuric chloride coupledmethoxypolyethylene glycol (mPEG) with protein of a prion, andpotentially with carbohydrate of a prion. All known prions includeprotein structure that can be similarly modified via the presentinvention with only slight variations in pH, temperature and time.Indeed, the pH, time and temperature conditions at which themodification reaction can be done at are very malleable, thus makingthis invention applicable to a wide variety of prion types. Otherpolymers may be utilized instead of mPEG, such as, inter alia,polyethylene glycol, ethoxypolyethylene glycol, dextran, ficoll, andarabinogalactan. Other linker molecules may be utilized instead ofcyanuric chloride, such as, inter alia, imidazolyl formate, succinimidylsuccinate, succinimidyl glutarate, N-hydroxysuccinimide, 4-Nitrophenol,2,4,5-trichlorophenol, and a chloroformate. FIG. 5 lists exemplarypolymeric linker compounds (PLCs) that may be used with the presentinvention and associated targets that can be reacted with the PLCs. Mostof the listed targets in FIG. 5 are proteins. The thiol groups in FIG. 5include sulfhydryl groups which are protein components. Any of the PLCsthat react with the hydroxyl group can be reacted with a carbohydrate.Note that the PLC of phospholipid PEG interacts with a lipid byintercalation rather than by covalent bonding.

[0045] As discussed supra, the present invention is based oninteractions between abnormal prions and normal prions. Suchinteractions may be understood in terms of protein-protein andcharge-charge coupling models, which are explained and confirmed asfollows.

[0046] Animal models have conclusively demonstrated that prion diseasecan be transmitted via food consumption or direct injection. The basicdisease process is dependent upon the interaction of the abnormal PrPprotein with the normal PrP protein (Scott et al. 1989; Scott et al.1993). This protein-protein interaction results in a conformationalchange in the normal PrP protein. Zahn et al. (2000) demonstrated thatin sheep the conformational transition to the scrapie form of PrP, whichcauses prion disease, is due to direct inter-molecular interactionsbetween the abnormal and normal PrP proteins. The effective transmissionof prion diseases depends on the abnormal prion being similar enough tothe host's normal prion protein to be able to ‘lock in’ to its structureand convert it. The disease pathogenesis does not require any geneticalteration in the infected animal.

[0047] As is well established in the scientific literature, all proteinshave an ‘optimal’ folding pattern that is mediated by intra-protein andinter-protein charge-charge interactions and amino acid hydrophobicityand hydrophilicity (Mirny et al., 2001). The ‘optimal’ shape is a stablelow energy state that is determined via the above hydrophobicity,hydrophilicity, and charge-charge interactions. The more hydrophobic(oily) an amino acid is, the less stable it is when surrounded by waterthus favoring the self-aggregation of hydrophobic residues and theexclusion of water while the hydrophilic residues fold so as to be incontact with water. This folding in turn is affected by the electricalcharge and the physical size of amino acids. All of these factorsbalance against each other until the protein twists into a stable, lowenergy state where the hydrophobic amino acids stabilize each other, andopposite intra-chain and/or inter-chain charges are close together(charge-charge interactions). It is further established that proteinsundergo significant conformational changes within cells both in vivo andin vitro. These changes include initial protein folding and subsequentunfolding, refolding, and transitional folding states that are inducedby intra-protein and inter-protein charge-charge interactions andsubstrate binding. These charge-charge interactions are in turninfluenced by the intra- and extracellular environment including pH,energy state, oxidant injury, and phosphorylation status. The optimalprotein structure can in turn be influenced by other proteins. Forexample, chaperone proteins are a class of proteins that trafficproteins within the cell, protect the protein against degradation, andassist in proper protein folding and refolding following biologicinsult. However, as with intra-chain folding, the protein-protein (e.g.,chaperone) mediated folding is also governed by charge-chargeinteraction and hyrophilic/hydrophobic interactions (Panse et al.,2000). Protein conformation can also be altered by substrate binding, aswell as protein phosphorylation, both of which altering the surfacecharge characteristics of a protein. An example of a substrate inducedconformnational change in a protein is seen in myosin which undergoes asignificant structural change following binding of adenosinetriphosphate (ATP) binding and ATP hydrolysis (Rayment, 1993).

[0048] While at least two distinct mechanisms have been proposed toaccount for an ability of the abnormal PrP protein to induce changes inthe normal protein, both mechanisms employ the same mechanisms ofprotein folding used to explain normal protein folding (Horiuchi et al.,1999; Prusiner, 1991; Jarrett and Lansbury, 1993; Cohen et al., 1994).Specifically, charge-charge, hydrophilic/hydrophobic, andprotein-protein changes in conformation are involved in PrP diseasepathogenesis. To accomplish this conformational change by the abnormalPrP protein, the coaggregation of the abnormal and normal PrP protein isa crucial event. This aggregation event allows for the close proximityof the abnormal and normal PrP protein and further enhances the relativeconcentration of the two proteins. Indeed, the relative concentration ofthe two proteins is important and is referred to as the nucleation(aggregation) event. Studies suggest that the initial nucleation(aggregation) event requires a critical mass (volume) of abnormal andnormal PrP protein (Horiuchi et al., 2000; Prusiner, 1991). In thisconformational change event, the abnormal PrP protein acts as a templatefor the refolding of the normal protein.

[0049] Hence, as described by the existing prion literature, a criticalevent in prion disease is the direct physical association of theabnormal PrP protein with the normal cellular homolog in this nucleation(aggregation) event (Horiuchi et al., 2000). This is showndiagrammatically in FIG. 1. Hence, prevention of this interaction couldprove beneficial in preventing or delaying the onset of prion-mediateddisease in human and nonhuman animals. The interaction of the abnormalprion protein can be prevented by agents that give rise to improvedsolubility (i.e., prevents aggregation of the protein) or prevents thephysical interaction of the abnormal and normal PrP proteins. Asdescribed in this invention, the covalent modification of the abnormalprion with polyethylene glycol (or its derivatives), dextran, orarabinogalactan, or any combination thereof as depicted in FIG. 5 anddiscussed supra, can prevent the conformation change of the normal PrPprotein. Said prevention of conformational change disables or removesthe “transforming power” of the abnormal prion as discussed supra inconjunction with FIG. 2. For example, derivatization of the abnormalprotein with methoxypoplyethylene glycol does the following: (1)improves solubility of the abnormal protein which diminishes theaggregation and critical nucleation event, as described by Horiuchi etal. (2000); and (2) prevents protein-protein interaction necessary forconformational conversion, as described by Prusiner (1991), Horiuchi etal. (2000), and others, by blocking charge-charge interactions as wellas sterically hindering the stable interaction of the abnormal andnormal PrP proteins. Furthermore, as shown in FIG. 6, one abnormal humanprotein associated with Creutzfeldt-Jakob Disease occurs as aconsequence of a single amino acid substitution of glutamate → lysine atcodon 200 (cited in some literature as codon 199) of a mutant human PrPgene/protein. The presence of this lysine group serves as a potentialtarget for a number of activated PEG agents as listed in FIG. 5 thusserving as an additional manner of preventing protein-proteininteraction. The sequence of normal and abnormal PrP proteins has beenelucidated and is highly amenable to covalent derivatization withactivated PEG species due to the presence of multiple lysine residues aswell as other targets for PEG-derivatization. See Stahl et al. (1993).

[0050] Biophysical, biochemical, and physiologic evidence that clearlyprovide foundational support this invention are summarized as follows.First as previously discussed, in the absence of a normal PrP protein(e.g., PrP knockout mice) the introduction of abnormal PrP does notinduce disease (Sakaguchiet al, 1996; Brander et al,. 1996). Hence,interaction of the abnormal and normal PrP proteins is essential fordisease pathogenesis. Second, as discussed supra, conformational changesin proteins are associated with protenaceous folding patterns, and saidfolding patterns result from intra-protein and inter-proteininteractions which are generally governed by charge-charge coupling andnucleation (aggregation) processes. Third, studies of pegylated proteinsand cells teach that pegylation camouflages the inherent charge ofproteins, thus preventing charge-charge associations. This is evidencedby the loss of movement in an electrical filed, which demonstrates theapparent loss of surface charge. See FIG. 7 (Scott and Bradley et al.,2000) discussed infra. Fourth, our studies demonstrate that pegylatedproteins and cells demonstrated reduced abilities to aggregate (i.e.,undergo nucleation events). See FIGS. 8 and 9 (Scott and Mahaney et al.,2000) discussed infra. Fifth, pegylated proteins and cells demonstrateenhanced solubility as evidenced by the improved solubility (i.e.,inability to sediment) of red blood cells (“RBC”) (FIG. 9; Scott andMahaney et al., 2000). These events of FIGS. 8 and 9 are evidenced bythe loss of Rouleaux formation (the stacking/aggregation of RBC) and thesubsequent loss of red blood cell sedimentation (settling). Similarly,studies of PEG-modified purified proteins and lipid particles alsoclearly demonstrated that their half-life within the circulation/tissueis significantly improved (see Herschfield, 1987; Beckman, 1988;Abuchowski, 1984; Zalipsky; 1994; Maruyama, 1992). Finally,receptor-ligand (a more complex protein-protein interaction) isprevented by all of the above events (Scott et al., 1998; Scott andBradley et al., 2000). As demonstrated by the above points, pegylationof proteins effectively prevents/diminishes the nucleation (aggregation)process necessary for prion disease pathogenesis. The precedingbiophysical, biochemical, and physiologic evidence provides foundationalsupport for the method and structure of the present invention asdepicted in FIG. 2.

[0051] In FIG. 7 from Scott and Bradley et al. (2000), covalentattachment of mPEG to the red blood cells (RBC) significantly reducesthe mobility of the cell in an applied electrical field. Loss ofmobility is mediated by both the camouflage of the inherent surfacecharge of the RBC and the increased drag placed on the cell by thecovalently attached mPEG polymers. Electrophoretic mobility measurementsof control and pegylated RBC were made on a Rank Mark I electrophoresisapparatus equipped with a horizontal microscope having a water immersionlens. Ten different RBC were chosen randomly and were manually timed todetermine their velocity in the field. The electrophoretic mobility wasdetermined as: mobility=velocity of the particle (μm/sec)/electric fieldstrength (E) in volt/cm; where E=(voltage/distance between theelectrodes). RBC's were covalently modified with benzotriazole carbonateof mPEG (“BTC-mPEG”) containing polymers of increasing molecular weight(3.4, 5, and 20 kDa). The RBC were covalently modified in isotonicsaline and washed 3-time in saline prior to electrophoretic mobilitydeterminations. The mPEG concentration is shown in millimoles of mPEGper liter (mMol/L) of the isotonic saline medium. At 20 kDa mPEG, a nullelectrophoretic mobility was achieved at an mPEG concentration of about1.2 mMol/L and higher, resulting in a total camouflaging of surfacecharge. At 5 kDa mPEG and 3.4 kDa mPEG, a partial camouflaging ofsurface charge was achieved at the highest mPEG concentrations,corresponding to a reduction of about a factor of 3.0 and 2.0,respectively in electrophoretic mobility. Thus, the camouflaging ofsurface charge becomes increasingly effective with increasing polymericmolecular weight and also with increasing mPEG concentration (until zeromobility is achieved).

[0052] In FIG. 8, pegylation of cells and proteins results in thecamouflage of protein associated charge. As a consequence of theobfuscation of protein charge, protein-mediated aggregation isprevented. This is evidenced by the loss of red blood cell RouleauxFormation (stacking) which is charge-mediated. As shown at top, normalred blood cells 40 form stacking structures 42 due to membrane surface(protein-protein) interactions. However, covalent attachment of mPEG 46to red blood cells 46, or its derivatives, camouflages the inherentcharge of the membrane surface proteins, which prevents the requiredprotein-protein interactions necessary for cell stacking and Rouleauxformation.

[0053] In FIG. 9, pegylation of cells and proteins results in thecamouflage of protein associated charge. This is evidenced by the lossof movement of particles in an electrical field as shown in FIG. 7 anddiscussed supra. Because of the obfuscation of protein charge,protein-mediated aggregation is prevented. This is evidenced by the lossof red blood cell Rouleaux Formation (stacking) which is charge-mediatedas shown in FIG. 8 and discussed supra. As shown in FIG. 9, normaluntreated red blood cells serve as a control and have a sedimentationrate of about 32 mm/hr. In contrast, consequent to the loss ofmicroaggregation, mPEG-modified red blood cells have a measuredsedimentation rate of only about 1 mm/hr, which is within the noiselevel of the measurement instrumentation. Thus, the mPEG-modified redblood cells no longer sediment and therefore remain soluble in solution(i.e., have improved solubility).

[0054] While particular embodiments of the present invention have beendescribed herein for purposes of illustration, many modifications andchanges will become apparent to those skilled in the art. Accordingly,the appended claims are intended to encompass all such modifications andchanges as fall within the true spirit and scope of this invention.

We claim:
 1. A method for forming a prion-physiological structure,comprising: providing an abnormal prion that has a transforming powerover a normal prion; and covalently bonding a linker molecule to theabnormal prion, wherein a polymer is covalently attached to the linkermolecule to form a polymerized abnormal prion that does not have thetransforming power over the normal prion.
 2. The method of claim 1,wherein the polymer is selected from the group consisting ofpolyethylene glycol, methoxypolyethylene glycol, ethoxypolyethyleneglycol, dextran, ficoll, and arabinogalactan.
 3. The method of claim 1,wherein the linker molecule is selected from the group consisting ofcyanuric chloride, imidazolyl formate, succinimidyl succinate,succinimidyl glutarate, N-hydroxysuccinimide, 4-Nitrophenol,2,4,5-trichlorophenol, and a chloroformate.
 4. The method of claim 1,wherein the abnormal prion has human significance.
 5. The method ofclaim 1, wherein the abnormal prion has veterinary significance.
 6. Themethod of claim 1, wherein covalently bonding the linker molecule to theabnormal prion includes covalently bonding the linker molecule to anamino acid at the abnormal prion.
 7. The method of claim 1, whereincovalently bonding the linker molecule to the abnormal prion includescovalently bonding the linker molecule to a lysine group at the abnormalprion.
 8. The method of claim 1, wherein covalently bonding the linkermolecule to the abnormal prion includes covalently bonding the linkermolecule to a carbohydrate at the abnormal prion.
 9. The method of claim1, wherein covalently bonding the linker molecule to the abnormal prionincludes covalently bonding the linker molecule to a sulfhydryl group atthe abnormal prion.
 10. A method for forming a prion-physiologicalstructure, comprising: providing an abnormal prion that has atransforming power over a normal prion; and covalently bonding a linkermolecule to the abnormal prion, wherein a polymer is covalently attachedto the linker molecule to form a polymerized abnormal prion, and whereinthe polymer has a long chain length that inactivates the transformingpower.
 11. A method for forming a prion-physiological structure,comprising: providing an abnormal prion that has a transforming powerover a normal prion; and covalently bonding a linker molecule to theabnormal prion, wherein a polymer is covalently attached to the linkermolecule to form a polymerized abnormal prion, and wherein acamouflaging by the polymer of a charge site at the abnormal prioninactivates the transforming power.
 12. A method for forming aprion-physiological structure, comprising: providing an abnormal prionthat has a transforming power over a normal prion; covalently bonding alinker molecule to the abnormal prion, wherein a polymer is covalentlyattached to the linker molecule to form a polymerized abnormal prionthat does not have the transforming power over the normal prion;providing an animal that includes the normal prion; and introducing thepolymerized abnormal prion into the animal.
 13. The method of claim 12,wherein the animal is a human animal.
 14. The method of claim 12,wherein the animal is a veterinary animal.
 15. A method for forming aprion-physiological structure, comprising: providing a medium thatincludes proteinaceous matter, said proteinaceous matter including anabnormal prion or not including the abnormal prion, said abnormal prionhaving a transforming power over a normal prion; providing a linkermolecule to which a polymer is covalently attached; and introducing thelinker molecule into the medium such that if the medium includes theabnormal prion then said introducing results in the linker moleculecovalently bonding to the abnormal prion to form a polymerized abnormalprion that does not have the transforming power over the normal prion.16. The method of claim 15, wherein the proteinaceous matter includesblood albumin.
 17. A method for forming a prion-physiological structure,comprising: providing a medium that includes proteinaceous matter, saidproteinaceous matter including an abnormal prion or not including theabnormal prion, said abnormal prion having a transforming power over anormal prion; providing a linker molecule to which a polymer iscovalently attached; introducing the linker molecule into the mediumsuch that if the medium includes the abnormal prion then saidintroducing results in the linker molecule covalently bonding to theabnormal prion to form a polymerized abnormal prion that does not havethe transforming power over the normal prion; providing an animal thatincludes the normal prion; and after introducing the linker molecule,introducing a portion of the medium into the animal.
 18. The method ofclaim 17, wherein the proteinaceous matter includes blood albumin. 19.The method of claim 17, wherein the animal is a human animal.
 20. Themethod of claim 17, wherein the animal is a veterinary animal.
 21. Aprion-physiological structure, comprising: an abnormal prion that has atransforming power over a normal prion; and a linker molecule covalentlybonded to the abnormal prion, wherein a polymer is covalently attachedto the linker molecule to form a polymerized abnormal prion that doesnot have the transforming power over the normal prion.
 22. Theprion-physiological structure of claim 21, wherein the polymer isselected from the group consisting of polyethylene glycol,methoxypolyethylene glycol, ethoxypolyethylene glycol, dextran, ficoll,and arabinogalactan.
 23. The prion-physiological structure of claim 21,wherein the linker molecule is selected from the group consisting ofcyanuric chloride, imidazolyl formate, succinimidyl succinate,succinimidyl glutarate, N-hydroxysuccinimide, 4-Nitrophenol,2,4,5-trichlorophenol, and a chloroformate.
 24. The prion-physiologicalstructure of claim 21, wherein the abnormal prion has humansignificance.
 25. The prion-physiological structure of claim 21, whereinthe abnormal prion has veterinary significance.
 26. Theprion-physiological structure of claim 21, wherein covalently bondingthe linker molecule to the abnormal prion includes covalently bondingthe linker molecule to an amino acid at the abnormal prion.
 27. Theprion-physiological structure of claim 21, wherein covalently bondingthe linker molecule to the abnormal prion includes covalently bondingthe linker molecule to a lysine group at the abnormal prion.
 28. Theprion-physiological structure of claim 21, wherein covalently bondingthe linker molecule to the abnormal prion includes covalently bondingthe linker molecule to a carbohydrate at the abnormal prion.
 29. Theprion-physiological structure of claim 21, wherein covalently bondingthe linker molecule to the abnormal prion includes covalently bondingthe linker molecule to a sulfhydryl group at the abnormal prion.
 30. Aprion-physiological structure, comprising: an abnormal prion that has atransforming power over a normal prion; and a linker molecule covalentlybonded to the abnormal prion, wherein a polymer is covalently attachedto the linker molecule to form a polymerized abnormal prion, and whereinthe polymer has a long chain length that inactivates the transformingpower.
 31. A prion-physiological structure, comprising: an abnormalprion that has a transforming power over a normal prion; and a linkermolecule covalently bonded to the abnormal prion, wherein a polymer iscovalently attached to the linker molecule to form a polymerizedabnormal prion, and wherein a camouflaging by the polymer of a chargesite at the abnormal prion inactivates the transforming power.
 32. Aprion-physiological structure, comprising: an abnormal prion that has atransforming power over a normal prion; a linker molecule covalentlybonded to the abnormal prion, wherein a polymer is covalently attachedto the linker molecule to form a polymerized abnormal prion that doesnot have the transforming power over the normal prion; and an animalthat includes the normal prion and the polymerized abnormal prion. 33.The prion-physiological structure of claim 32, wherein the animal is ahuman animal.
 34. The prion-physiological structure of claim 32, whereinthe animal is a veterinary animal.
 35. A prion-physiological structure,comprising a medium having: proteinaceous matter that includes anabnormal prion or does not include the abnormal prion, wherein theabnormal prion has a transforming power over a normal prion; and alinker molecule to which a polymer is covalently attached, wherein ifthe medium includes the abnormal prion then the linker molecule iscovalently bonded to the abnormal prion to form a polymerized abnormalprion that does not have the transforming power over the normal prion.36. The prion-physiological structure of claim 35, wherein theproteinaceous matter includes blood albumin.
 37. A prion-physiologicalstructure, comprising an animal that includes a normal prion and aportion of a medium, wherein the medium comprises: proteinaceous matterthat includes an abnormal prion or does not include the abnormal prion,wherein the abnormal prion has a transforming power over the normalprion; and a linker molecule to which a polymer is covalently attached,wherein if the medium includes the abnormal prion then the linkermolecule is covalently bonded to the abnormal prion to form apolymerized abnormal prion that does not have the transforming powerover the normal prion.
 38. The prion-physiological structure of claim37, wherein the proteinaceous matter includes blood albumin.
 39. Theprion-physiological structure of claim 37, wherein the animal is a humananimal.
 40. The prion-physiological structure of claim 37, wherein theanimal is a veterinary animal.
 41. A prion-physiological structure,comprising: an abnormal prion that has a transforming power over anormal prion; and means for covalently bonding a linker molecule to theabnormal prion, wherein a polymer is covalently attached to the linkermolecule to form a polymerized abnormal prion that does not have thetransforming power over the normal prion.
 42. The prion-physiologicalstructure of claim 41, wherein the polymer is selected from the groupconsisting of polyethylene glycol, methoxypolyethylene glycol,ethoxypolyethylene glycol, dextran, ficoll, and arabinogalactan.
 43. Theprion-physiological structure of claim 41, wherein the linker moleculeis selected from the group consisting of cyanuric chloride, imidazolylformate, succinimidyl succinate, succinimidyl glutarate,N-hydroxysuccinimide, 4-Nitrophenol, 2,4,5-trichlorophenol, and achloroformate.
 44. The prion-physiological structure of claim 41,wherein the abnormal prion has human significance.
 45. Theprion-physiological structure of claim 41, wherein the abnormal prionhas veterinary significance.
 46. The prion-physiological structure ofclaim 41, wherein covalently bonding the linker molecule to the abnormalprion includes covalently bonding the linker molecule to an amino acidat the abnormal prion.
 47. The prion-physiological structure of claim41, wherein covalently bonding the linker molecule to the abnormal prionincludes covalently bonding the linker molecule to a lysine group at theabnormal prion.
 48. The prion-physiological structure of claim 41,wherein covalently bonding the linker molecule to the abnormal prionincludes covalently bonding the linker molecule to a lysine group at theabnormal prion.
 49. The prion-physiological structure of claim 41,wherein covalently bonding the linker molecule to the abnormal prionincludes covalently bonding the linker molecule to a sulfhydryl group atthe abnormal prion.
 50. A prion-physiological structure, comprising: anabnormal prion that has a transforming power over a normal prion; andmeans for covalently bonding a linker molecule to the abnormal prion,wherein a polymer is covalently attached to the linker molecule to forma polymerized abnormal prion, and wherein the polymer has a long chainlength that inactivates the transforming power.
 51. Aprion-physiological structure, comprising: an abnormal prion that has atransforming power over a normal prion; and means for covalently bondinga linker molecule to the abnormal prion, wherein a polymer is covalentlyattached to the linker molecule to form a polymerized abnormal prion,and wherein a camouflaging by the polymer of a charge site at theabnormal prion inactivates the transforming power.
 52. Aprion-physiological structure, comprising: an abnormal prion that has atransforming power over a normal prion; means for covalently bonding alinker molecule to the abnormal prion, wherein a polymer is covalentlyattached to the linker molecule to form a polymerized abnormal prionthat does not have the transforming power over the normal prion; ananimal that includes the normal prion; and means for introducing thepolymerized abnormal prion into the animal.
 53. The prion-physiologicalstructure of claim 52, wherein the animal is a human animal.
 54. Theprion-physiological structure of claim 52, wherein the animal is aveterinary animal.
 55. A prion-physiological structure, comprising: amedium that includes proteinaceous matter, said proteinaceous matterincluding an abnormal prion or not including the abnormal prion, saidabnormal prion having a transforming power over a normal prion; a linkermolecule to which a polymer is covalently attached; and means forintroducing the linker molecule into the medium such that if the mediumincludes the abnormal prion then said introducing results in the linkermolecule covalently bonding to the abnormal prion to form a polymerizedabnormal prion that does not have the transforming power over the normalprion.
 56. The prion-physiological structure of claim 55, wherein theproteinaceous matter includes blood albumin.
 57. A prion-physiologicalstructure, comprising: a medium that includes proteinaceous matter, saidproteinaceous matter including an abnormal prion or not including theabnormal prion, said abnormal prion having a transforming power over anormal prion; a linker molecule to which a polymer is covalentlyattached; means for introducing the linker molecule into the medium suchthat if the medium includes the abnormal prion then said introducingresults in the linker molecule covalently bonding to the abnormal prionto form a polymerized abnormal prion that does not have the transformingpower over the normal prion; an animal that includes the normal prion;and means for introducing a portion of the medium into the animal. 58.The prion-physiological structure of claim 57, wherein the proteinaceousmatter includes blood albumin.
 59. The prion-physiological structure ofclaim 57, wherein the animal is a human animal.
 60. Theprion-physiological structure of claim 57, wherein the animal is aveterinary animal.